CN1869066A - Tumour antigen protein and tumour antigen peptide - Google Patents

Abstract

The present invention relates to a tumor antigen protein and tumor antigen peptide. The tumor antigen protein can be decomposed in cells to produce peptide fragments combined with major histocompatibility complex (MHC)-class I molecules and recognized by T cells. The DNA sequence encoding the tumor antigen protein has the nucleotide sequence of the sequence whose accession number is TSPYNM 003308 in the gene library. The tumor antigen protein and tumor antigen peptide can be used to prepare medicine for treating liver cancer.

Description

A kind of tumor antigen protein and tumor antigen peptide

technical field

The invention relates to a tumor antigen protein and a tumor antigen peptide, in particular to a tumor antigen protein and a tumor antigen peptide for treating liver cancer.

Background technique

The occurrence of tumor is related to the immune state of the body. Cancer patients are often accompanied by hypofunction of cellular immunity, and cellular immunity plays a major role in clearing tumors. The immunogenicity of tumors is weak and cannot induce the body to produce antibodies sufficient to resist tumors. Some tumor products have an immunosuppressive effect on the host, so it is difficult for the immune system of tumor patients to launch a powerful attack on the tumor. Anticancer drugs are generally immunosuppressants, which reduce the immunity of patients while treating tumors. Radiotherapy and chemotherapy are not only difficult to cure tumors but also lead to the occurrence of second tumors. Therefore, in the treatment of malignant tumors, how to improve the immune function of patients has become the key to the success of malignant tumor treatment.

It is known that the immune system in the human body, including humoral immunity and cellular immunity, especially T cell immunity, plays an important role in killing and inhibiting tumors. With the understanding of the nature of the immune response, people found that no matter how complicated the process of the immune response is, what ultimately leads to the activation of T cells and the killing effect is to combine with the major histocompatibility complex MHC to form antigen peptide complexes and co- Stimulatory signal, that is, cytotoxic T cells (CTL) recognize tumor antigen proteins/peptides and major histocompatibility complex (MHC) class I antigens expressed on the surface of tumor cells through T cell receptors (TCR) on their own surface Combined to form a complex to attack and kill tumor cells.

The production of tumor antigen protein/peptide is firstly expressed by tumor cells as tumor antigen, which is decomposed into antigen peptides with a size of 8-10 amino acids by various proteases in the cells. These antigen peptides and MHC class I produced in the endoplasmic reticulum Molecular binding is expressed on the surface of cells through the Golgi complex, and finally gives tumor antigen-specific CTLs, thereby inducing an immune response and killing and clearing tumor cells. This protein antigen molecule that exists in tumor cells and can bind to MHC molecules and induce the body's immune response is a tumor antigen. These antigens can be used to induce the body to produce an immune response that only targets tumor cells, thereby specifically killing tumor cells in the body to achieve the effect of treating or preventing tumor cell metastasis. In recent decades, people have focused on the search for tumor-specific and/or related antigens in the treatment of tumors with immune methods.

In the 1950s, Foley et al. immunized mice with chemically induced tumors with inactivated autologous tumor cells, and then inoculated the mice with live tumor cells, which were rejected and unable to grow Mice survived when they formed tumors; while mice that were not immunized with inactivated autotumor cells were inoculated with live tumor cells, the tumor cells were not rejected and grew into tumors, and the mice died. This experiment proved convincingly that there are tumor-specific antigens in chemically induced tumors, but this protective response has precise specificity, that is, it is only against the same type of tumor and not against different types of tumors, even for Different types of tumors induced in the same mouse with the same carcinogen were also not protective.

In 1991, T. Boon et al. identified the tumor antigen protein from human melanoma cells for the first time and named it MAGE (Science, 254:1643-1647, 1991). Subsequently, other tumor antigen proteins were identified, which can be divided into five categories: tumor-testis (CT) antigen protein, differentiation antigen protein, mutated tumor antigen protein, overexpressed tumor antigen protein and virus antigen protein. Among them, tumor-testis (CT) antigen protein is the most widely used clinically.

Tumor-testis (CT) antigen proteins are the most identified class of tumor antigens, and their encoding genes are characterized by their expression in many types of tumors, such as melanoma, lung cancer, sarcoma and bladder cancer It is expressed, but it is not expressed in normal tissues except the testis, and there is a certain amount of expression in the placenta, ovary, and pancreas. Because the testis is an immune privileged site, this type of antigen is considered to be a tumor-specific antigen in therapy, and it is the most promising type of antigen for tumor immunotherapy. At present, this type of antigen is mainly used clinically, such as MAGE-1 and NY-ESO-1, which have a high positive rate in certain types of tumors and have good immunogenicity. , has a good prospect when used in tumor immunotherapy.

So far, the most representative methods for identifying tumor antigen proteins are: library screening based on specific cellular immune response, cDNA expression library screening based on humoral immune response, combinatorial peptide library screening, pickling Detachment method, bioinformatics method and cDNA microarray method.

Liver cancer is the most common and malignant tumor among human tumors, especially in recent years, the incidence rate has gradually increased. Due to the high degree of malignancy and rapid growth of liver cancer, once it is discovered, it is already in the middle and late stage. At present, the treatment of liver cancer is mainly local treatment methods such as traditional surgical resection and radiotherapy. However, since most patients have entered the middle and late stage when they are diagnosed, And liver cancer is quite resistant to radiotherapy and chemotherapy. Therefore, the treatment effect and prognosis are often poor, and the one-year and five-year survival rates of liver cancer patients are very low compared with the survival rates of other types of tumor patients. Finding breakthroughs in treatment methods or new treatment methods has always been the goal pursued by clinical oncologists. People are looking forward to new methods to improve the early diagnosis of liver cancer and the good prognosis of patients, as well as to remove residual tumor cells in the body after surgery. and prevent tumor cell metastasis.

Contents of the invention

The purpose of the present invention is to provide a tumor antigen protein and tumor antigen peptide which can be used for liver cancer treatment.

The purpose of the present invention is achieved through the following technical solutions:

The present invention provides a tumor antigen protein, which can produce a peptide fragment combined with major histocompatibility complex (MHC)-class I molecules and recognized by T cells through intracellular decomposition, which has the following characteristics: The amino acid sequence described in (a) or (b):

(a) having the amino acid sequence shown in Sequence 1;

(b) A derivative protein produced by substituting, deleting or adding mutations to the amino acid sequence in (a), and the derivative protein has the same function as the protein in (a).

The present invention provides a DNA sequence, which encodes said tumor antigen protein.

The DNA sequence of the present invention has the nucleotide sequence shown in Sequence 2, and the accession number in the gene library is: TSPY NM 003308.

The invention provides the application of said protein as tumor-testis antigen.

The invention provides the application of the gene as tumor-testis antigen gene.

The present invention provides a tumor antigen peptide having the same function as the peptide fragment produced by the tumor antigen protein, which has the amino acid sequence of the 70th to 78th amino acid sequence in sequence 1.

The application of the tumor antigen protein of the present invention in the preparation of medicines for treating liver cancer.

The application of the tumor antigen peptide of the present invention in the preparation of drugs for treating liver cancer.

The advantages of the tumor antigen protein and tumor antigen peptide provided by the present invention are: the present invention identifies the TSPY gene as a tumor-testis antigen gene for the first time; when immunotherapy is used to treat cancer, these antigens can be used to induce the body to produce tumor-specific antigens. The immune response of cells, while not killing normal cells, specifically kills tumor cells in the body to achieve the effect of treating or preventing tumor cell metastasis; at the same time, the occurrence and development of tumors can be detected by detecting the tumor antigen gene or antibody. And whether there is metastasis for diagnosis.

Description of drawings

Fig. 1 is the RT-PCR detection result of the gene TSPY encoding the antigenic protein of the present invention in 16 kinds of adult normal tissues; 1～16 in the figure represent respectively adult normal tissues spleen, prostate, ovary, small intestine, large intestine, white blood cells, heart, lung , liver, brain, kidney, placenta, testis, skeletal muscle, pancreas, thymus.

Fig. 2 is the RT-PCR result of the gene TSPY encoding the antigenic protein of the present invention in the cancer tissue of liver cancer and the paired paracancerous tissue; T in the figure represents the cancerous tissue, and N represents the paired paracancerous tissue.

Figure 3 is the result of in situ hybridization of TSPY in liver cancer tissue, where A is the experimental group and B is the sense probe control group.

Fig. 4 is the representative result of detecting the recombinant protein of antigenic protein of the present invention in Escherichia coli and the serum reaction of liver cancer patient by Western blot method; 1 in the figure represents protein molecular weight standard reference, and 2-15 represent expresses the tumor in the present invention - The results of the reaction of the E. coli lysate of the testis antigen recombinant protein with the serum of liver cancer patients, 16-17 represent the results of the reaction of the E. coli lysate expressing the tumor-testis antigen recombinant protein of the present invention with normal human serum.

Detailed ways

The tumor antigen protein and tumor antigen peptide provided by the present invention will be described in more detail below through examples.

Embodiment 1, cDNA microarray (cDNA microarray) analysis

In order to find the genes differentially expressed in liver cancer tissues and adjacent tissues, we analyzed the differential expression of more than 18,000 genes in liver cancer tissues and adjacent tissues using the commercially available AFFYMETRIX human genome U-133A chip. In the data analysis, we found that a testis-specific gene TSPY is highly expressed in liver cancer tissues. The accession number of the TSPY gene in the gene library is TSPY NM 003308, which has the nucleotide sequence shown in Sequence 2.

Embodiment 2, analyze the expression of TSPY gene with the method for RT-PCR

cDNA of 16 commercial human normal tissues from the biotechnology company CLONTECH (spleen, prostate, testis, ovary, small intestine, large intestine, white blood cells, heart, lung, liver, brain, kidney, pancreas, placenta, skeletal muscle, thymus), cDNA from cancer tissues and paired paracancerous tissues of 57 liver cancer patients, cDNA from 19 renal cancer tissues and paired paracancerous tissues, and cancer tissues and paired paracancerous tissues of 13 lung cancer patients The cDNA of TSPY gene, the cDNA from cancer tissue and paired paracancerous tissue of 10 patients with gastric cancer, and the cDNA of cancer tissue and paired paracancerous tissue from 8 cases of bladder cancer were used to detect the expression of TSPY gene in normal tissue and tumor expression of the organization.

Conditions for RT-PCR in the present invention: DNA polymerase from Shenergy Bocai Co., Ltd., 94°C, 20 seconds; 58°C, 20 seconds; 72°C, 20 seconds; after 32 cycles, 72°C, 7 minutes. The primer sequence for RT-PCR, forward primer: CAGGGCTTCTCATTCCACTC; reverse primer: CCATCATATTCAACTCAACAACTGG. It was found that: TSPY gene is only expressed in normal testis, and not expressed in other normal human tissues (as shown in Figure 1); None of the paracancerous tissues expressed (as shown in Figure 2).

Embodiment 3, in situ hybridization

In order to confirm the cell localization of TSPY and further prove the correlation between the gene and liver cancer, in situ hybridization was carried out with paraffin sections of liver cancer tissues.

The PCR product amplified by the upstream primer CAGGGCTTCTCATTCCACTC and the downstream primer CCATCATATTCAACTCAACAACTGG was constructed into a plasmid (the standard plasmid in Real time PCR), and the sequence was confirmed to be correct by sequencing. Sense and antisense RNA probes were labeled with DIG RNA labeling Kit (T7/SP6) (Roche) according to the kit instructions.

The brief operation steps are as follows: Prepare 6 μm thick paraffin sections, after dewaxing and hydration treatment, treat with 0.2M hydrochloric acid at room temperature for 10 minutes, wash with PBS for 2×5 seconds, digest with 20 μg/ml proteinase K at 37°C for 30 minutes . Wash 2 x 5 sec with PBS and fix with 4% paraformaldehyde for 10 min. Wash with PBS for 3 x 5 seconds, dehydrate with 70%, 95%, 100% gradient ethanol. Add 30 μl prehybridization solution (50% formamide, 10% dextran sulfate, 1×Denhard's solution, 20mM Tris-HCl (pH 8.0), 300mM NaCl, 1mM EDTA, 250μg/ml yeast tRNA) dropwise to each section, and pre-hybridize at 50℃. Hybridization was performed for 60 minutes. Then, hybridization solution containing 0.5 μg/ml probe was used for hybridization at 50° C. for 16 hours. After hybridization, wash with 2×SSC, 50% formamide at 37°C for 30 minutes, then wash with 2×SSC at 37°C for 2×15 minutes, and 0.1×SSC at 37°C for 2×15 minutes. Hybridization signals were detected with an alkaline phosphatase-conjugated digoxigenin-labeled antibody (1:500 dilution). The blue-purple signal is a positive signal.

Results The expression of TSPY was detected in the detected hepatocellular carcinoma cells, but not in the adjacent tissues. However, no positive hybridization signal was seen in hybridization with the sense control probe (as shown in FIG. 3 ).

Embodiment 4, the expression of TSPY gene recombinant protein in escherichia coli

Using the pQE30 prokaryotic expression vector (INVOTRIGEN), IPTG induced the expression of the recombinant protein for 6 hours in Escherichia coli strain M15 (INVOTRIGEN) under the growth condition of 37°C. The expression of recombinant protein was identified by SDS-PAGE protein electrophoresis. Afterwards, the recombinant protein was separated and purified from the whole protein of E. coli using a nickel ion affinity chromatography column (INVOTRIGEN). The recombinant protein was identified by using the monoclonal antibody against the 6 histidines contained in the recombinant protein by Western hybridization.

Embodiment 5, detect the antibody of TSPY recombinant protein in the serum of liver cancer patient by Western hybridization method

With reference to the method in Journal of Experimental Medicine, 187:1349, 1998, the serum of tumor patients with positive TSPY mRNA transcript was screened, and it was found that antibodies against TSPY recombinant protein existed in the serum of tumor patients (Fig. 4). Therefore, TSPY is a new tumor-testis antigen, which has the ability to induce immune response in tumor patients and kill tumor cells, and has the potential to be applied to clinical immunotherapy of tumors.

Example 6. Application of Tumor Antigen Peptides to Induce CTL Formation in Vitro and CTL Killing Experiments in Vitro

Refer to the experimental method in Chinese Journal of General Surgery (17: 218, 2002). Culture 2ml of PBMC (peripheral blood mononuclear cells) at a concentration of 2× ^{10 6 /} ml in a 24-well culture plate as effector lymphocytes. Add tumor antigen peptide 40ug/ml to the base, incubate at 37°C for 2 hours, irradiate with 30GY ⁶⁰ Co gamma rays, centrifuge, wash off the remaining peptide, and use as antigen cells, the number of APC cells is 1.3:1 according to the effector cells: APC, Antigen cells were added to the above-mentioned effector cells for co-culture, and IL-2 (provided by Ludwig Institute for Cancer Research, Australia) 25U/ml was added after 24 hours to continue the culture. After that, at intervals of 7 days, the antigen-presenting cells were added to the effector cells according to the effector cell:antigen cell ratio of 1.3:1, and the antigen-presenting cells were stimulated three times, and the killing effect of CTL was detected on the 28th day. The killing experiment was carried out as follows: (1) Using a non-radioactive cytotoxicity assay box, measuring the LDH release value after CTL and target cells were co-cultured to detect the killing effect of CTL. Operate according to the recommended procedure of the kit. The medium used was phenol red-free RPMI 1640 containing 3% FCS. Three replicate wells were set up for each reaction, and the spontaneous release value of target cells, the maximum release value of target cells, the spontaneous release value of effector cells, the volume correction value and Medium background value. The following formula was used to calculate the killing effect: killing effect (%)=(experimental value-effector cell spontaneous release value-target cell spontaneous release value)/(target cell maximum release value-target cell spontaneous release value)×100. (2) After the mixed lymphocytes and target cells were co-cultured for 4 hours, the morphological changes of lymphocytes and target cells were observed. After 28 days, peripheral blood mononuclear cells expanded 32-fold, among which the proportion of CD3 positive cells increased by 18% (from 50% to 68%), and the proportion of CTL increased by 20% (from 32% to 52%). When the ratio of effector cells: target cells was 10:1, the killing effect of CTL on autologous lymphoblastocytes incubated with tumor antigen peptide (synthesized by Shanghai Gil Biochemical Co., Ltd.) was 52.5%, and the killing effect on tumor cells positive for tumor antigen TSPY The effect was 36.2%, both of which were significantly higher than the killing effect on autologous lymphoblastoid cells (15.8%) and the killing effect on tumor cells negative for tumor antigen TSPY (1.6%); when the ratio of effector cells: target cells was 3.3:1 , the killing effect of CTL on autologous lymphoblastocytes incubated with tumor antigen peptide was 52.3%, which was significantly higher than the killing effect on autologous lymphoblastocytes (14.3%). These results indicate that the tumor antigen peptide of tumor antigen TSPY can effectively induce CTL with specific killing tumor cells from the PBMC of tumor patients, thereby enhancing the ability of tumor patients to clear tumors.

Example 7. Detection of CTL cells secreting IFNγ ability after being stimulated by tumor antigen peptide

Refer to the experimental method in Chinese Medical Journal (81: 1234, 2001). Quantification of IFN-γ was performed by enzyme-linked immunospot assay (ELISPOT). 96-well flat-bottomed nitrocellulose plate (purchased from Millipore, France), coated with anti-human IFNγ monoclonal antibody (dissolved in pH 9.6 carbonate buffer, final concentration 2ug/ml), overnight at 4°C; After being washed with phosphate buffered saline (PBS) containing 0.05% Tween 20, blocked with RPMI 1640 culture solution containing 10% AB serum for 1 hour at room temperature in the dark, washed with 0.05% Tween PBS, and added to the 7-day-induced Effector cells (Effector cells, E) 5×10 ⁴ per well. Afterwards, the experiment was divided into two groups, that is, the effector cell and T2 cell group (E+T2), and the effector cell and T2 cell loading tumor antigen peptide group (E+T2 tumor antigen peptide), and each group made duplicate wells. Pure T2 cells and T2 cells loaded with 10ug/ml tumor antigen peptide were incubated in serum-free RPMI 1640 for 2 hours at 37°C and 5% CO ₂ , irradiated with ⁶⁰ Coγ rays 100GY, and washed with serum-free RPMI 1640 to remove excess After the antigen peptide was used as stimulator cells, 1×10 ⁵ cells/well were added to each effector cell well; the reaction system was incubated in RPMI 1640 culture medium without cytokines and serum at 37°C and 5% CO ₂ for 18 ~20 hours later, wash the culture plate with 0.05% TweenPBS to remove residual cells; then add 0.2ug/ml biotin-labeled anti-human IFNγ secondary antibody, incubate at 37°C for 2 hours, wash the plate; add alkaline phosphatase labeling Streptavidin 1ug/ml, incubate for 1 hour at room temperature in the dark; wash the plate, add the substrate chromogenic solution, develop color in the dark for 5 minutes, rinse with tap water, and terminate the reaction. After the reaction plate was dried, count the number of black-purple spots in each well under a dissecting microscope, and calculate the average value of the duplicate wells. The number of spots in the E+T2 tumor antigen peptide group minus the number of spots in the E+T2 group was the frequency of tumor antigen-specific CTL cells. In ELISPOT detection, using the frequency of IFNγ-producing CD8 ⁺ T cells as an index, the responders to the tumor antigen TSPY were detected in patients whose tumor tissue expressed TSPY mRNA, but were not detected in patients without TSPY mRNA expression in cancer tissue to the corresponding specific immune response. In tumor patients whose tumor tissues express TSPY mRNA but have no serum TSPY antibody, the immune response of CD8+ T cells to tumor antigen TSPY and antigenic peptide can still be detected. This experiment provides a basis for the further application of tumor antigen TSPY and antigenic peptide in clinical in vivo experiments to treat tumors.

The application of the tumor antigen protein and tumor antigen peptide provided by the present invention can provide medicine for activating anti-tumor immunity and a method for diagnosing tumors. The antibody against the tumor antigen protein or tumor antigen peptide of the present invention can be used in affinity chromatography, screening of cDNA library, immunological diagnosis or preparation of medicine.

sequence listing

Sequence 1 is the amino acid sequence of tumor-testis antigen protein TSPY

Sequence length: 308

Topology: Linear

Sequence Type: Peptide

source:

Biological name: Human (Homo sapiens)

  Tissue Type: Liver Cancer Tissue

Sequence features:

Signs of features: peptides

Existence location: 1..183

sequence 1;

Met Arg Pro Glu Gly Ser Leu Thr Tyr Arg Val Pro Glu Arg Leu Arg

1 5 10 15

Gln Gly Phe Cys Gly Val Gly Arg Ala Ala Gln Ala Leu Val Cys Ala

20 25 30

Ser Ala Lys Glu Gly Thr Ala Phe Arg Met Glu Ala Val Gln Glu Gly

35 40 45

Ala Ala Gly Val Glu Ser Glu Gln Ala Ala Leu Gly Glu Glu Ala Val

50 55 60

Leu Leu Leu Asp Asp Ile Met Ala Glu Val Glu Val Val Ala Glu Glu

65 70 75 80

Glu Gly Leu Val Glu Arg Arg Glu Glu Ala Gln Pro Arg Gln Gln Ala

85 90 95

Val Pro Gly Pro Gly Pro Met Thr Pro Glu Ser Ala Leu Glu Glu Leu

100 105 110

Leu Ala Val Gln Val Glu Leu Glu Pro Val Asn Ala Gln Ala Arg Lys

115 120 125

Ala Phe Ser Arg Gln Arg Glu Lys Met Glu Arg Arg Arg Lys Pro His

130 135 140

Leu Asp Arg Arg Gly Ala Val Ile Gln Ser Val Pro Gly Phe Trp Ala

145 150 155 160

Asn Val Ile Ala Asn His Pro Gln Met Ser Ala Leu Ile Thr Asp Glu

165 170 175

Asp Glu Asp Met Leu Ser Tyr Met Val Ser Leu Glu Val Glu Glu Glu Glu

180 185 190

Lys His Pro Val His Leu Cys Lys Ile Met Leu Phe Phe Arg Ser Asn

195 200 205

Pro Tyr Phe Gln Asn Lys Val Ile Thr Lys Glu Tyr Leu Val Asn Ile

210 215 220

Thr Glu Tyr Arg Ala Ser His Ser Thr Pro Ile Glu Trp Tyr Pro Asp

225 230 235 240

Tyr Glu Val Glu Ala Tyr Arg Arg Arg His His Asn Ser Ser Leu Asn

245 250 255

Phe Phe Asn Trp Phe Ser Asp His Asn Phe Ala Gly Ser Asn Lys Ile

260 265 270

Ala Glu Ile Leu Cys Lys Asp Leu Trp Arg Asn Pro Leu Gln Tyr Tyr

275 280 285

Lys Arg Met Lys Pro Pro Glu Glu Gly Thr Glu Thr Ser Gly Asp Ser

290 295 300

Gln Leu Leu Ser

305

Sequence 2 is the nucleotide sequence of tumor-testis antigen protein gene TSPY

Sequence length: 1159

Chain type: double chain

Topology: Linear

Sequence type: cDNA to mRNA

Hypothetical sequence: none

Antonym: None

origin:

Biological name: Human (Homo sapiens)

  Tissue Type: Liver Cancer Tissue

Sequence features:

Characteristic notation: 5’UTR

Existence position: 1..45

Characteristic notation: CDS

Existence location: 46..972

Characteristic notation: 3’UTR

Existence location: 973..1159

Characteristic notation: polyA site

Existence location: 1096..1137

Sequence 2:

ggcccttcgc gcgcagtccc ttagggggcg cctggaagcc cgcgcatgcg ccctgagggc 60

tcgctgacct accgggtgcc agagaggctg cggcagggtt tctgtggcgt gggtcgggca 120

gcacaggcct tggtgtgtgc gagtgccaag gagggcaccg ccttcaggat ggaggctgtg 180

caggaggggg cggccggggt ggagagtgag caggcggctt tggggggagga ggcggtgctg 240

ctgttggatg acataatggc ggaggtggag gtggtggcgg aggaggggg cctcgtggag 300

cggcgggagg aggcccagcc ccgacagcag gctgtgcctg gccctgggcc catgacccca 360

gagtctgcac tggaggagct gctggccgtt caggtggagc tggagccggt taatgcccaa 420

gccaggaagg ccttttctcg gcagcgggaa aagatggagc ggaggcgcaa gccccaccta 480

gaccgcagag gcgccgtcat ccagagcgtc cctggcttct gggccaatgt tattgcaaac 540

cacccccaga tgtcagccct gatcactgac gaagatgaag acatgctgag ctacatggtc 600

agcctggagg tggaagaaga gaagcatcct gttcatctct gcaagatcat gttgttcttt 660

cggagtaacc cctacttcca gaataaagtg attaccaagg aatatctggt gaacatcaca 720

gaatacagggg cttctcattc cactccaatt gagtggtatc cggattatga agtggaggcc 780

tatcgccgca gacaccacaa cagcagcctt aacttcttca actggttctc tgaccacaac 840

ttcgcaggat ctaacaagat tgctgagatc ctatgtaagg acctgtggcg caatcccctg 900

caatactaca agaggatgaa gccacctgaa gagggaacag agacgtcagg ggactcccag 960

ttgttgagtt gaatatgatg gagcatcaga ttttacctaa tacagcagaa ctcctaaaaa 1020

gttacagcca tatgcaggac ggcagtactc agcatggtct tatgcacagg aactaaagga 1080

aaaagagatc gagtcacaaa aattcaggaa gagggggtaa atgtggattg tatggaatga 1140

aaaataaaca ttctcaagg 1159

SEQUENCE LISTING

<110> Peking University

<120> A tumor antigen protein and tumor antigen peptide

<130>FPI05111

<160>2

<170>PatentIn version 3.1

<210>1

<211>308

<212>PRT

<213>Homo sapiens

<400>1

Met Arg Pro Glu Gly Ser Leu Thr Tyr Arg Val Pro Glu Arg Leu Arg

1 5 10 15

Gln Gly Phe Cys Gly Val Gly Arg Ala Ala Gln Ala Leu Val Cys Ala

20 25 30

Ser Ala Lys Glu Gly Thr Ala Phe Arg Met Glu Ala Val Gln Glu Gly

35 40 45

Ala Ala Gly Val Glu Ser Glu Gln Ala Ala Leu Gly Glu Glu Ala Val

50 55 60

Leu Leu Leu Asp Asp Ile Met Ala Glu Val Glu Val Val Ala Glu Glu

65 70 75 80

Glu Gly Leu Val Glu Arg Arg Glu Glu Ala Gln Pro Arg Gln Gln Ala

85 90 95

Val Pro Gly Pro Gly Pro Met Thr Pro Glu Ser Ala Leu Glu Glu Leu

100 105 110

Leu Ala Val Gln Val Glu Leu Glu Pro Val Asn Ala Gln Ala Arg Lys

115 120 125

Ala Phe Ser Arg Gln Arg Glu Lys Met Glu Arg Arg Arg Lys Pro His

130 135 140

Leu Asp Arg Arg Gly Ala Val Ile Gln Ser Val Pro Gly Phe Trp Ala

145 150 155 160

Asn Val Ile Ala Asn His Pro Gln Met Ser Ala Leu Ile Thr Asp Glu

165 170 175

Asp Glu Asp Met Leu Ser Tyr Met Val Ser Leu Glu Val Glu Glu Glu Glu

180 185 190

Lys His Pro Val His Leu Cys Lys Ile Met Leu Phe Phe Arg Ser Asn

195 200 205

Pro Tyr Phe Gln Asn Lys Val Ile Thr Lys Glu Tyr Leu Val Asn Ile

210 215 220

Thr Glu Tyr Arg Ala Ser His Ser Thr Pro Ile Glu Trp Tyr Pro Asp

225 230 235 240

Tyr Glu Val Glu Ala Tyr Arg Arg Arg His His Asn Ser Ser Leu Asn

245 250 255

Phe Phe Asn Trp Phe Ser Asp His Asn Phe Ala Gly Ser Asn Lys Ile

260 265 270

Ala Glu Ile Leu Cys Lys Asp Leu Trp Arg Asn Pro Leu Gln Tyr Tyr

275 280 285

Lys Arg Met Lys Pro Pro Glu Glu Gly Thr Glu Thr Ser Gly Asp Ser

290 295 300

Gln Leu Leu Ser

305

<210>2

<211>1159

<212>DNA

<213>Homo sapiens

<400>2

ggcccttcgc gcgcagtccc ttagggggcg cctggaagcc cgcgcatgcg ccctgagggc 60

tcgctgacct accgggtgcc agagaggctg cggcagggtt tctgtggcgt gggtcgggca 120

gcacaggcct tggtgtgtgc gagtgccaag gagggcaccg ccttcaggat ggaggctgtg 180

caggaggggg cggccggggt ggagagtgag caggcggctt tggggggagga ggcggtgctg 240

ctgttggatg acataatggc ggaggtggag gtggtggcgg aggaggggg cctcgtggag 300

cggcgggagg aggcccagcc ccgacagcag gctgtgcctg gccctgggcc catgacccca 360

gagtctgcac tggaggagct gctggccgtt caggtggagc tggagccggt taatgcccaa 420

gccaggaagg ccttttctcg gcagcgggaa aagatggagc ggaggcgcaa gccccaccta 480

gaccgcagag gcgccgtcat ccagagcgtc cctggcttct gggccaatgt tattgcaaac 540

cacccccaga tgtcagccct gatcactgac gaagatgaag acatgctgag ctacatggtc 600

agcctggagg tggaagaaga gaagcatcct gttcatctct gcaagatcat gttgttcttt 660

cggagtaacc cctacttcca gaataaagtg attaccaagg aatatctggt gaacatcaca 720

gaatacagggg cttctcattc cactccaatt gagtggtatc cggattatga agtggaggcc 780

tatcgccgca gacaccacaa cagcagcctt aacttcttca actggttctc tgaccacaac 840

ttcgcaggat ctaacaagat tgctgagatc ctatgtaagg acctgtggcg caatcccctg 900

caatactaca agaggatgaa gccacctgaa gagggaacag agacgtcagg ggactcccag 960

ttgttgagtt gaatatgatg gagcatcaga ttttacctaa tacagcagaa ctcctaaaaa 1020

gttacagcca tatgcaggac ggcagtactc agcatggtct tatgcacagg aactaaagga 1080

aaaagagatc gagtcacaaa aattcaggaa gagggggtaa atgtggattg tatggaatga 1140

aaaataaaca ttctcaagg 1159

Claims (8)

1, a kind of tumor antigen protein matter, this protein is by decomposing, can produce the peptide fragment that combines with major histocompatibility complex (MHC)-I quasi-molecule, can be discerned by the T cell in the cell, and it has following (a) or (b) described aminoacid sequence:

(a) has the aminoacid sequence shown in the sequence 1;

(b) with the aminoacid sequence in (a) through the replacement of one or several amino-acid residue, disappearance or add the derived protein that sudden change is produced, and this derived protein has identical functions with (a) protein.

2, the described tumor antigen protein matter of a kind of claim 1 is as the application of tumor-testis antigen.

3, the application of the described tumor antigen protein matter of a kind of claim 1 in preparation treatment liver-cancer medicine.

4, a kind of dna sequence dna, the described tumor antigen protein matter of its coding claim 1.

5, dna sequence dna as claimed in claim 4 is characterized in that: described its is to have the nucleotide sequence shown in the sequence 2, and its accession number in gene library is TSPY NM 003308.

6, the described gene of a kind of claim 5 is as the application of tumor-testis antigen gene.

7, the tumor antigen peptide of the peptide fragment same function that produced of the described tumor antigen protein matter of a kind of and claim 1, it has the 70th～the 78th aminoacid sequence of aminoacid sequence in the sequence 1.

8, the application of the described tumor antigen peptide of a kind of claim 7 in preparation treatment liver-cancer medicine.

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